Post on 10-May-2015
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RECOMBINANT THERAPEUTIC PROTEINS
ATRACTIVE FEATURES
• Therapeutic proteins are used for the treatment of abnormal health conditions.
• Replacing chemically synthesized drugs• Highly specific function• less interference with normal biological processes • faster clinical development and FDA approval time • less likely to elicit immune responses.
Evolution of Protein Therapeutic
1982 Human insulin, created using recombinant DNA technology
1986 Interferon alfa and muromonab-CD3 approved 1993 CBER's Office of Therapeutics Research and
Review (OTRR) formed 1997 First whole chimeric antibody, rituximab, and
first humanized antibody, daclizumab, approved
2002 $30 billion share of biotechnological drugs of $400 billion in yearly worldwide pharmaceutical sales
2006 An inhaled form of insulin (Exubera) approved
Classification of Protein therapeutics
• Group I: protein therapeutics with enzymatic or regulatory activity
• Group II : protein therapeutics with special targeting activity
• Group III : protein vaccines• Group IV : protein diagnostics
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Process of Drug Production
Cells and plasmid
+
Cell line
Transfection Cell culture Purification
Drug substance (crude)
Drug substance (pure)
Drug product -(sterile)
Formulation/Filling
Cell line manufactureMedium development
Bioreactor process development & scale-up
Downstream purification
Analytical characterization
World market
- EPO alone : ~ $ 11
Billion per year
- - $ 50 Billion
(2007) $ 190
Billion (2015)
- Antibodies > 50
%
- Intensive
investment in
monoclonal
antibodies:
Therapeutic proteins will form the back-born of future medicinal therapy
PRODUCTION SYSTEMS FOR PRODUCING THERAPEUTIC PROTEINS:
• simple physiology • short generation times, as bacteria grow and multiply
rapidly • large yields of product - up to 10 % of mass (low cost)• The expressed proteins often do not fold properly and
so are biologically inactive.• The synthesised protein is often toxic to bacteria
preventing the cell cultures from reaching high densities
BACTERIAL CELLS
Yeast cells
• Yeast is a simple eukaryote and performs many of the post-translational modifications required for human proteins
• Presence of active proteases that degrade foreign (expressed) proteins, thereby reducing their yield
• (a solution to this problem is the construction of yeast strains from which the protease genes have been deleted).
Bacillus Sp.
Actinomycetes Sp.
Eschericia coli
(Yeast)
MICROBES USED FOR PROTEIN PRODUCTION
• High level of expression • Correct folding
• More difficult to work with • Expensive
• Slow generation time • Not suitable for proteins with repetitive sequences
INSECT CELLS
• competitive cost • the availability of established practices for their
efficient harvesting, transporting, • sorting and processing
PLANTS
Transgenic animals
• cheap method • produce the desired proteins in vast quantities when
using larger animals like cows.• long lead time to generate a herd of transgenic
animals • concerns about the health of the animal. • cause serious negative health effects
In vitro systems• E. coli extract; plant wheatgerm extract; and mammalian sources, rabbit reticulocyte lysate.
• lack both the genomic material and the cellular boundary system
• contain the cellular components required for transcription and/or translation of genes.
Comparison of Recombinant Protein Expression
PURIFICATION STEPS
Some recombinant proteins approved for human use
Protein Company Disorder
Factor VIII Baxter, Bayer Hemophilia A
Factor IX Genetics Institute Hemophilia B
Tissue plasminogen activator (TPA)
Genetech Acute myocardial infarction
Insulin Eli Lilly, Novo Nordisk Diabetes mellitus
Human growth hormone
Eli Lilly, Genetech, Upjohn, Novo Nordisk
GH deficiency in children (dwarfism)
Erythropoietin Amgen, Ortho Biotech Anemia
DNase I Genetech Cystic fibrosis
Various interferons (IFN)
Schering, Biogen, Chiron,Genetech
Hepatitis B and C, multiple sclerosis
PRODUCTION OF RTPs
DRUG ANIMAL USED GENETIC MODIFICIATION
WHO/WHERE PRODUCED
Anti-Cancer Drugs (currently in the
process of making).
Chickens The anti-cancer drug is produced in the chickens eggs.
Roslin Institute in the United Kingdom is
LACTOFERRIN (Breast Milk Supplement)
COWS Recombinant DNA targets lysosome from the breast
milk and modifies it in the cow which
gives a more nutritional boost
for infants
Hermitech, Inc. in Sioux Falls, South
Dakota.& China
Drugs in transgenic animals
Production of Recombinant Therapeutic Proteins in the Milk of
Transgenic Animals
Schematic representation of the process used to
purify ATryn from the milk of transgenic goats.
Recombinant Hepatitis vaccine
• The hepatitis B virus (HBV) vaccine– surface antigen purified from the
blood of infected individuals. – Due to safety concerns, the HBV
vaccine became the first to be produced using recombinant DNA technology (1986)
– Produced in bakers’ yeast (Saccharomyces cerevisiae
• to respond to a human influenza pandemic.•
to respond to a human influenza pandemic.
Vaccine Production at industry level
FOLLICLE STIMULATING HORMONE (FSH)
• single vector containing the coding sequences for both subunit genes.
• After transfection, a genetically stable transformant producing biologically active recombinant FSH was isolated
• 150–450 gene copies were present in mammalian cells.
• FSH products from cell culture supernatants.
• collected from a perfusion-type bioreactor
• downstream purification
rhDNase I
CANCER VACCINE
Bio therapeutics are delicate drugs
• Much larger and more complex than traditional pharmaceuticals
• Composed of unstable proteins with a precise structure
• Easily damaged by unfavorable temperature history during storage